Fumigatus Hyphae Research Articles

Tracking the uptake of labelled host-derived extracellular vesicles by the human fungal pathogen Aspergillus fumigatus

Abstract Extracellular vesicles (EVs) have gained attention as facilitators of intercellular as well as interkingdom communication during host-microbe interactions. Recently we showed that upon infection, host polymorphonuclear leukocytes produce antifungal EVs targeting the clinically important fungal pathogen Aspergillus fumigatus; however, the small size of EVs (< 1 µm) complicates their functional analysis. Here, we employed a more tractable, reporter-based system to label host alveolar epithelial cell-derived EVs and enabled their visualisation during in vitro A. fumigatus interaction. Fusion of EV marker proteins (CD63, CD9, and CD81) with a Nanoluciferase (NLuc) and a green fluorescent protein (GFP) facilitated their relative quantification by luminescence and visualization by a fluorescence signal. The use of an NLuc fused with a GFP is advantageous as it allows for quantification and visualisation of EVs simultaneously without additional external manipulation and to distinguish subpopulations of EVs. Using this system, visualisation and tracking of EVs was possible using confocal laser scanning microscopy and advanced imaging analysis. These experiments revealed the propensity of host cell-derived EVs to associate with the fungal cell wall and ultimately colocalize with the cell membrane of A. fumigatus hyphae in large numbers. In conclusion, we have created a series of tools to better define the complex interplay of host-derived EVs with microbial pathogens.

Glucocorticoids Suppress NF-κB-Mediated Neutrophil Control of Aspergillus fumigatus Hyphal Growth.

Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. In this article, we used a zebrafish larva Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection, and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. Dexamethasone partially suppresses NF-κB activation at the infection site by inducing the transcription of IκB via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKKγ to prevent NF-κB activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKKγ crispant larvae further increases invasive hyphal growth and host mortality, suggesting that dexamethasone may suppress other pathways in addition to NF-κB to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-κB-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae.

Highly effective inhalable voriconazole-loaded nanomicelles for fungal infections in cystic fibrosis patients: A promising therapeutic strategy for allergic bronchopulmonary aspergillosis

In patients with cystic fibrosis (CF), growth of A. fumigatus and other fungal hyphae within the bronchial lumen can trigger an immunoglobulin E-mediated hypersensitivity response known as allergic bronchopulmonary aspergillosis (ABPA) which can potentially advance to fibrosis.The management of ABPA involves the administration of systemic glucocorticoids alongside azole antifungals, such as voriconazole (VRC). This study evaluated the potential of Soluplus® polymeric micelles as an effective nanocarrier for pulmonary delivery of VRC to improve antifungal therapy in CF patients. The micellar nanoformulation prepared in this study could increase up to 51-fold the aqueous solubility of VRC. Their micellar size, evaluated by dynamic light scattering, was 102.2 ± 1.3 nm for loaded freeze-dried nanoformulations with a unimodal size distribution. Nanomicelles showed excellent colloidal stability during the in vitro nebulization process and under dilution in similar interstitial fluids for pulmonary VRC delivery. Nanoformulations demonstrated high in vitro cytocompatibility in A549 and Vero cells. Interestingly, in vitro antifungal activity of VRC-loaded micelles was at least 2-fold higher than the drug aqueous suspension against different fungal species isolated from CF sputum, regardless of the presence of artificial CF mucus layer. Following single intratracheal dose, significantly (p < 0.05) higher VRC concentrations were observed within 2 h in the lung tissue for the micellar formulations compared with the VRC suspension. We concluded that these findings will help to develop a potent and innovative inhalable VRC micellar nanoformulation that may be effective against different lung disorders caused by fungi, such as ABPA.

Beyond the Charge: Interplay of Nanogels' Functional Group and Zeta-Potential for Antifungal Drug Delivery to Human Pathogenic Fungus Aspergillus Fumigatus.

The ubiquitous mold Aspergillus fumigatus (A. fumigatus) is one of the main fungal pathogens causing invasive infections in immunocompromised humans. Conventional antifungal agents exhibit limited efficacy and often cause severe side effects. Nanoparticle-based antifungal delivery provides a promising alternative, which can increase local drug concentration; while, mitigating toxicity, thereby enhancing treatment efficacy. Previous research underscores the potential of poly(glycidol)-based nanogels (NG) with negative surface charge as carriers for delivering antifungals to A. fumigatus hyphae. In this study, NG is tailored with 2-carboxyethyl acrylate (CEA) or with phosphoric acid 2-hydroxyethyl acrylate (PHA). It is discovered that quenching with PHA clearly improves the adhesion of NG to hyphal surface and the internalization of NG into the hyphae under protein-rich conditions, surpassing the outcomes of non-quenched and CEA-quenched NG. This enhancement cannot be solely attributed to an increase in negative surface charge but appears to be contingent on the functional group of the quencher. Further, it is demonstrated that itraconazole-loaded, PHA-functionalized nanogels (NGxPHA-ITZ) show lower MIC in vitro and superior therapeutic effect in vivo against A. fumigatus compared to pure itraconazole. This confirms NGxPHA as a promising antifungal delivery system.

Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography-Mass Spectrometry.

The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can be used to identify compounds interacting with the enzymes of isoprenoid biosynthesis, an important part of the ergosterol biosynthesis pathway. The method was validated according to the EMEA guideline on bioanalytical method validation. Aspergillus fumigatus hyphae and Saccharomyces cerevisiae cells were lysed mechanically in an aqueous buffer optimized for the enzymatic deconjugation of isoprenoid pyrophosphates. The residual alcohols were extracted, silylated and analyzed by GC-MS. The obtained isoprenoid pattern provides an indication of the inhibited enzyme, due to the accumulation of specific substrates. By analyzing terbinafine-treated A. fumigatus and mutant strains containing tunable gene copies of erg9 or erg1, respectively, the method was verified. Downregulation of erg9 resulted in a high accumulation of intracellular farnesol as well as elevated levels of geranylgeraniol and isoprenol. The decreased expression of erg1 as well as terbinafine treatment led to an increased squalene content. Additional analysis of growth medium revealed high farnesyl pyrophosphate levels extruded during erg9 downregulation.

Innate and Adaptive Immune Responses Induced by Aspergillus fumigatus Conidia and Hyphae.

Previous research indicated that hyphae of Aspergillus fumigatus (A. fumigatus) rather than conidia could successfully build a pulmonary aspergillosis model in immunocompetent mice. In this study, we compared the immune responses induced by hyphae and conidia to explore the possible mechanism of this striking phenomenon. Herein, a novel method was designed and adopted to quantify hyphal fragments. Murine macrophages RAW264.7 and human peripheral blood mononuclear cells were stimulated by A. fumigatus hyphae and conidia in vitro, respectively, and then immunological reactions were measured. Male C57BL/6 mice were challenged with conidia and hyphae through intratracheal inoculation. Dynamic conditions of mice were recorded, and RNA-seq measured corresponding immune responses. The results of the study confirmed that hyphae could induce more intensive inflammation than conidia in vitro and in vivo. However, macrophages revealed a higher production of ROS and M1 polarisation in response to conidia stimuli. Additionally, conidia could promote Th1 cell differentiation, while hyphae could increase the CD4/CD8 ratio. RNA-seq validated the fact that those multiple immunologically relevant pathways were more strongly activated by hyphae than conidia, which also promoted Th2 cell differentiation and suppressed Th1 signalling. Both hyphae and conidia could activate Th17 signalling. In general, conidia and hyphae induced distinctly different host immune responses, and the immune responses induced by conidia played a better protective effect. Therefore, the unique function of hyphae in the spread and infection of Aspergillus should be emphasised, and more research is required to clarify the underlying mechanisms for better understanding and management of aspergillosis.

S8.3c Cell wall glycans as targets for the development of new antifungals

S8.3 How the fungal cell wall glycan can modulate the immune response?, September 23, 2022, 3:00 PM - 4:30 PMBeing an extracellular organelle, the cell wall plays a crucial role in fungal life, by protecting fungal cells from a hostile environments and providing cells with mechanical strength. Exploiting the essentiality of this cell wall, available antifungal drugs target cell wall biosynthesis directly or by altering the fungal cell membrane. However, the emergence of resistance to antifungal drugs, new at risk-cohorts, and drug-drug interaction issue with antifungals demand new/alternative therapeutic strategy. In this regard, we have identified that surfactant protein-D (SP-D; a host humoral immune component) has growth inhibitory activity on Aspergillus fumigatus, a ubiquitous airborne opportunistic pathogen. SP-D, a soluble pattern recognition receptor of the collection family, targets galactosaminogalactan and galactomannan, the cell wall glycans of A. fumigatus hyphae, as the ligands. Hyphae grown in presence of SP-D show a significant decrease in the growth and are hyperbranched compared to control hyphae. SP-D treatment alters surface exposure of hyphal cell wall polysaccharides, thereby modifying hyphal immunoreactivity. SP-D pre-treatment increases the efficacy of the echinocandin and azole classes of antifungals against A. fumigatus. Interestingly, SP-D show hyphal growth inhibitory activity against multi-/pan-azole-resistant isolates of A. fumigatus. Overall, SP-D seems to target A. fumigatus cell wall glycans to execute its fungistatic activity. We are currently investigating the exact mechanism of anti-A. fumigatus activity associated with SP-D.

Invasive Pulmonary Aspergillosis with Marked Eosinophilia as a Unique Presentation of Chronic Granulomatous Disease

BackgroundChronic granulomatous disease (CGD) is a rare immunodeficiency disorder resulting in phagocytic cell dysfunction. It is characterized by deficient cellular immunity against bacteria and fungi, and an excessive inflammatory response resulting in granuloma formation. It manifests, usually in early childhood, with recurrent bacterial and fungal infections or inflammatory complications. The infections, such as invasive pulmonary aspergillosis, can be life-threatening.Case descriptionOur patient was a 40-year-old man with no pulmonary history who presented with bilateral pulmonary nodules and pronounced eosinophilia in peripheral blood and bronchoalveolar lavage fluid, mimicking eosinophilic pneumonia. During treatment with corticosteroids, the patient deteriorated clinically and radiographically. Extensive investigations failed to provide a diagnosis. A lung biopsy demonstrated the presence of granulomas and Aspergillus fumigatus hyphae. Advanced screening to detect underlying immunodeficiency revealed CGD.DiscussionThis case report describes a unique first presentation of CGD. It reminds physicians of the possibility of CGD as an underlying immune disorder in invasive aspergillosis and highlights the challenges of diagnosing invasive pulmonary aspergillosis. We discuss the diagnostic pitfalls of this case and propose a diagnostic work-up for eosinophilic lung disease.LEARNING POINTSPulmonary aspergillosis can present as eosinophilic pneumonia and should be included in the differential diagnosis of eosinophilic lung disease.In case of invasive pulmonary aspergillosis, investigation for chronic granulomatous disease should be considered.Chronic granulomatous disease in adults is probably underdiagnosed because of its variable clinical presentations.

Co-Operative Biofilm Interactions between Aspergillus fumigatus and Pseudomonas aeruginosa through Secreted Galactosaminogalactan Exopolysaccharide.

The mold Aspergillus fumigatus and bacterium Pseudomonas aeruginosa form biofilms in the airways of individuals with cystic fibrosis. Biofilm formation by A. fumigatus depends on the self-produced cationic exopolysaccharide galactosaminogalactan (GAG), while P. aeruginosa biofilms can contain the cationic exopolysaccharide Pel. GAG and Pel are rendered cationic by deacetylation mediated by either the secreted deacetylase Agd3 (A. fumigatus) or the periplasmic deacetylase PelA (P. aeruginosa). Given the similarities between these polymers, the potential for biofilm interactions between these organisms were investigated. P. aeruginosa were observed to adhere to A. fumigatus hyphae in a GAG-dependent manner and to GAG-coated coverslips of A. fumigatus biofilms. In biofilm adherence assays, incubation of P. aeruginosa with A. fumigatus culture supernatants containing de-N-acetylated GAG augmented the formation of adherent P. aeruginosa biofilms, increasing protection against killing by the antibiotic colistin. Fluorescence microscopy demonstrated incorporation of GAG within P. aeruginosa biofilms, suggesting that GAG can serve as an alternate biofilm exopolysaccharide for this bacterium. In contrast, Pel-containing bacterial culture supernatants only augmented the formation of adherent A. fumigatus biofilms when antifungal inhibitory molecules were removed. This study demonstrates biofilm interaction via exopolysaccharides as a potential mechanism of co-operation between these organisms in chronic lung disease.

Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice.

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.

Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy

Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.

Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus.

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

Antifungal Siderophore Conjugates for Theranostic Applications in Invasive Pulmonary Aspergillosis Using Low-Molecular TAFC Scaffolds.

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose-limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus (A. fumigatus), with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. A variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5) with antifungal activity were coupled to diacetylfusarinine C (DAFC), resulting in a “Trojan horse” to deliver antifungal compounds specifically into A. fumigatus hyphae by the major facilitator transporter MirB. Radioactive labeling with gallium-68 allowed us to perform in vitro characterization (distribution coefficient, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds chelated with stable gallium were used for antifungal susceptibility tests. [Ga]DAFC-fludioxonil, -FOA, and -Cy5 revealed a MirB-dependent active uptake with fungal growth inhibition at 16 µg/mL after 24 h. Visualization of an A. fumigatus infection in lungs of a rat was possible with gallium-68-labeled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. Overall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET imaging combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by A. fumigatus.

Production of egg yolk antibody against A.fumigatus and its therapeutic potential for treating A.fumigatus keratitis

ObjectiveTo prepare specific IgY against A. fumigatus and verify its specificity and antifungal effect on A. fumigatus keratitis. MethodLay hens were immunized with the suspension of inactivated A. fumigatus hyphae which mixed with Freund's complete adjuvant or incomplete Freund's adjuvant. The IgY protein specific for A. fumigatus was extracted by ammonium sulfate salting-out method at the fifth to eighth week after immunization. Bradford method and indirect ELISA were used to determine the concentration and titer of IgY. To verify the inhibitory effect of specific IgY on fungal growth, 1 × 105 CFU/mL A. fumigatus hyphae suspension and specific IgY of different concentrations were mixed and cultured for 24 h, 48 h and 72 h to measure the absorbance. Using specific IgY to treat A. fumigatus keratitis in mice, we observed the cornea under a slit lamp at 24 h, 72 h, and 120 h after treatment. Clinical score was used to assess the disease severity of fungal keratitis in mice cornea. The indirect ELISA method was used to determine the titer of specific IgY stored at room temperature and 4 °C for 1, 2, 3, 4, 5, 6, 7, 8, and 9 months. ResultsThe protein concentrations of specific IgY at the fifth, sixth, seventh and eighth weeks after immunization were 5.46 mg/mL, 5.79 mg/mL, 26.98 mg/mL, 28.71 mg/mL. The titer of the specific IgY of A. fumigatus can reach 1:10000, and the antifungal effect of the specific IgY is dose dependent within a certain range. Specific IgY treatment alleviated the severity of fungal keratitis of mice and reduced the clinical score. Moreover, there were no significant change in the titer of specific IgY after storage at room temperature for 2 months and storage at 4 °C for 6 months. ConclusionThe specific IgY can be successfully prepared by ammonium sulfate salting-out method. And it has excellent stability and significant antifungal effect on A. fumigatus keratitis.

Allicin modulates expression of cell wall-related genes in Aspergillus fumigatus

Background: Aspergillus fumigatus causes life-threatening iinvasive aspergillosis, especially in immunocompromised hosts, and resistance of this filamentous fungus to commonly used antifungal agents has been on the rise. Allicin (diallyl-dithiosulfinate), an allyl-sulfur compound from garlic has been proven to exhibit antimicrobial properties. However, there have been few studies on the antifungal mechanism of allicin against A. fumigatus. Based on our previous studies on the antifungal mechanism of allicin against Candida albicans, an opportunistic yeast pathogen, we hypothesize that allicin would similarly target cell-wall biosynthesis and integrity in A. fumigatus. This study evaluated the expression of cell-wall related genes in A. fumigatus ATCC 36607 upon treatment with allicin. Methods and materials: The minimum inhibitory concentration (MIC) of allicin against A. fumigatus ATCC 36607was determined according to the CLSI M38-A2 guidelines. A. fumigatus was incubated in the presence of allicin at subinhibitory concentrations of 1/4X MIC, 1/2X MIC, and 1X MIC for 48 hours. The cells were harvested post-incubation with allicin and RNA was extracted and reverse-transcribed into cDNA for evaluation of expression of selected genes via real time PCR. Results: The minimum MIC of allicin against A. fumigatus was found to be 3.2 μg/ml. Scanning electron micrographs exhibited significant morphological alteration of A. fumigatus hyphae and conidia after 48 h incubation at 1× MIC, 2× MIC and sub-MIC concentrations of allicin 1/4× MIC and 1/2× MIC in comparison with untreated (growth) control. Upon treatment at 1/4× MIC (0.8 μg/ml), hyphae were seen as wrinkled with patchy and rough surface implying decrease in structural integrity. Further, at 1/2× MIC (1.6 μg/ml), aberrant shape of hyphae with irregular constriction was observed along the length of hypha. At 1X MIC, complete suppression of hyphae growth was observed and only conidia were visible. Real-time PCR findings revealed that significant down-regulation of chsE, chsG, fksA, and gel2 genes were observed at various MICs of allicin tested. On the contrary, rhoD showed an upregulated expression. Conclusion: This study suggests the ability of allicin to affect the expression of cell wall-related genes of A. fumigatus.

Aspergillus fumigatus-Stimulated Human Corneal Epithelial Cells Induce Pyroptosis of THP-1 Macrophages by Secreting TSLP.

Fungal keratitis (FK) is a keratopathy caused by pathogenic fungal infection. The aim of this work is to explore the role of thymic stromal lymphopoietin (TSLP) in FK. Human corneal epithelial cells (HCECs) were treated with Aspergillus fumigatus hyphae, and we found that TSLP was highly expressed and secreted in the hyphae-treated HCECs. Hyphae-treated HCECs or TSLP treatment enhanced the expression of caspase-1 P20, GSDMD-N (p30), IL-1β, and IL-18 in the human THP-1 macrophages. The influence conferred by hyphae-treated HCECs or TSLP treatment was rescued by TSLP neutralizing antibody or VX-765 (caspase-1 inhibitor) treatment. Moreover, TSLP treatment promoted the expression of NLRP3, ASC, caspase-1 P20, GSDMD-N (p30), IL-1β, and IL-18 in the THP-1 macrophages, which was abolished by NLRP3 knockdown. Furthermore, TSLPR silencing suppressed the expression of NLRP3, ASC, caspase-1 P20, GSDMD-N (p30), IL-1β, and IL-18 in the TSLP-treated THP-1 macrophages. In conclusion, our article confirms that Aspergillus fumigatus-stimulated HCECs induce pyroptosis of THP-1 macrophages by secreting TSLP. TSLP/TSLPR induces caspase-1-dependent pyroptosis through activation of NLRP3 inflammasome. Thus, our work suggests that TSLP may be a potential target for FK treatment.

Immune Cell Paracrine Signaling Drives the Neutrophil Response to A. fumigatus in an Infection-on-a-Chip Model.

Neutrophils act as first responders during an infection, following signals from the pathogen as well as other host cells to migrate from blood vessels to the site of infection. This tightly regulated process is critical for pathogen clearance and, in many cases, eliminates the pathogen without the need for an additional immune response. It is, therefore, critical to understand what signals drive neutrophil migration to infection in a physiologically relevant environment. In this study, we used an infection-on-a-chip model to recapitulate many important aspects of the infectious microenvironment including an endothelial blood vessel, an extracellular matrix, and the environmental fungal pathogen Aspergillus fumigatus. We then used this model to visualize the innate immune response to fungal infection. We found that A. fumigatus germination dynamics are influenced by the presence of an endothelial lumen. Furthermore, we demonstrated that neutrophils are recruited to and swarm around A. fumigatus hyphae and that the presence of monocytes significantly increases the neutrophil response to A. fumigatus. Using secreted protein analysis and blocking antibodies, we found that this increased migration is likely due to signaling by MIP-1 family proteins. Finally, we demonstrated that signal relay between neutrophils, mediated by LTB4 signaling, is also important for sustained neutrophil migration and swarming in response to A. fumigatus infection in our system. Taken together, these results suggest that paracrine signaling from both monocytes and neutrophils plays an important role in driving the neutrophil response to A. fumigatus.

The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus.

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5′-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae.

Multi trace element profiling in pathogenic and non-pathogenic fungi

Maintaining appropriate levels of trace elements during infection of a host is essential for microbial pathogenicity. Here we compared the uptake of 10 trace elements from 3 commonly-used laboratory media by 3 pathogens, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, and a model yeast, Saccharomyces cerevisiae. The trace element composition of the yeasts, C. albicans, C. neoformans and S. cerevisiae, grown in rich (YPD) medium, differed primarily in P, S, Fe, Zn and Co. Speciation analysis of the intracellular fraction, which indicates the size of the organic ligands with which trace elements are complexed, showed that the ligands for S were similar in the three fungi but there were significant differences in binding partners for Fe and Zn between C. neoformans and S.cerevisiae. The profile for Cu varied across the 3 yeast species. In a comparison of C. albicans and A. fumigatus hyphae, the former showed higher Fe, Cu, Zn and Mn, while A. fumigatus contained higher P, S Ca and Mo. Washing C. albicans cells with the cell-impermeable chelator, EGTA, depleted 50–90 % of cellular Ca, suggesting that a large proportion of this cation is stored in the cell wall. Treatment with the cell wall stressor, Calcofluor White (CFW), alone had little effect on the elemental profile whilst combined Ca + CFW stress resulted in high cellular Cu and very high Ca. Together our data enhance our understanding of trace element uptake by pathogenic fungi and provide evidence for the cell wall as an important storage organelle for Ca.

Potential role of N-acetyl glucosamine in Aspergillus fumigatus-assisted Chlorella pyrenoidosa harvesting

BackgroundAlgal harvesting is a major cost which increases biofuel production cost. Algal biofuels are widely studied as third-generation biofuel. However, they are yet not viable because of its high production cost which is majorly contributed by energy-intensive biomass harvesting techniques. Biological harvesting method like fungal-assisted harvesting of microalgae is highly efficient but poses a challenge due to its slow kinetics and poorly understood mechanism.ResultsIn this study, we investigate Aspergillus fumigatus–Chlorella pyrenoidosa attachment resulting in a harvesting efficiency of 90% within 4 h. To pinpoint the role of extracellular metabolite, several experiments were performed by eliminating the C. pyrenoidosa or A. fumigatus spent medium from the C. pyrenoidosa–A. fumigatus mixture. In the absence of A. fumigatus spent medium, the harvesting efficiency dropped to 20% compared to > 90% in the control, which was regained after addition of A. fumigatus spent medium. Different treatments of A. fumigatus spent medium showed drop in harvesting efficiency after periodate treatment (≤ 20%) and methanol–chloroform extraction (≤ 20%), indicating the role of sugar-like moiety. HR-LC–MS (high-resolution liquid chromatography–mass spectrometry) results confirmed the presence of N-acetyl-d-glucosamine (GlcNAc) and glucose in the spent medium. When GlcNAc was used as a replacement of A. fumigatus spent medium for harvesting studies, the harvesting process was significantly faster (p < 0.05) till 4 h compared to that with glucose. Further experiments indicated that metabolically active A. fumigatus produced GlcNAc from glucose. Concanavalin A staining and FTIR (Fourier transform infrared spectroscopy) analysis of A. fumigatus spent medium- as well as GlcNAc-incubated C. pyrenoidosa cells suggested the presence of GlcNAc on its cell surface indicated by dark red dots and GlcNAc-specific peaks, while no such characteristic dots or peaks were observed in normal C. pyrenoidosa cells. HR-TEM (High-resolution Transmission electron microscopy) showed the formation of serrated edges on the C. pyrenoidosa cell surface after treatment with A. fumigatus spent medium or GlcNAc, while Atomic force microscopy (AFM) showed an increase in roughness of the C. pyrenoidosa cells surface upon incubation with A. fumigatus spent medium.ConclusionsResults strongly suggest that GlcNAc present in A. fumigatus spent medium induces surface changes in C. pyrenoidosa cells that mediate the attachment to A. fumigatus hyphae. Thus, this study provides a better understanding of the A. fumigatus-assisted C. pyrenoidosa harvesting process.